CLIN. CHEM. 30/5, 707-711 (1984)
Quantification of Pepsin A Activity in Canine and Rat Gastric Juice with the
Chromogenic Substrate Azocoll
P. C. Will, W. E. Allbee, C. G. Wftt,R. J. Bertko, and 1. S Gaglnella1
The activity concentration of pepsin may be quantified by
using azocoll as a chromogenic substrate.The measured
enzyme activity
isconstant between pH 1.2 and 3.4 and is
proportional (r = 0.61) to the activity
measured withhemoglobin as substrate. The activity of purified porcine pepsin is
inhibited by pepstatin A with an apparent K( of 115 nmol/L.
The azocoll method is useful for measuring changes in
pepsin secretion in response to pharmacological agents. For
example, pepsin activity ofcanine gastric juice is decreased
by 80% after in vivo administration of 0.5 mg of the synthetic
trimethyl prostanoid Ro 22-6923 per kilogram of body weight.
The method issufficiently
sensitiveto measure the pepsin
activity
in0.2L ofcaninegastric
juicewitha CV of -10%, is
simpler
than
the hemoglobin-substrate
methods,
and the
substrate is commercially available.
AddItIonal
activity
Keyphrases:
prostaglandin
Materials
Azocoll (50-100 mesh) and purified porcine pepsin (cat.
no. 516360) were purchased from Calbiochem-Behring,
San
Diego, CA 92112. Citricacid,bovine hemoglobin (type H),
and pepstatinA were from Sigma Chemical Co., St. Louis,
MO 63178. All other reagents were purchased from various
suppliers as ACS-certifled grade or better. Flat-bottom 96well microtiter plates (Immulon 2) were obtained from
Dynatech
Laboratories, Inc., Alexandria,
VA 22314. The
synthetic
trimethyl
prostanoid
Ro 22-6923 was obtained
from Dr. G. W. Holland, Hoffmann-La
Roche Inc., Nutley,
NJ 07110.
GastricJuice Specimens
.
pepstatin
.
enzyme
The proteolytic enzyme pepsin A (EC 3.4.23.1) issecreted
as the pepsinogenby thechiefcellsofthegastricmucosa of
vertebrates(1). An endopeptidase with an aspartic acid
residue at the active site (2, 3), the enzyme is inhibited
by
the pentapeptide pepstatin A (4) and the polycyclic phenol
gossypol(5). Pepsin activity has been determined with use of
various substrates such as hemoglobin (6), azo-albumin (7,
8), and synthetic peptides (2, 3), with which the activity is
proportional to the appearance of amino acids (2, 6), dye (7,
8), or other products (3, 9). Several of these assays require
that the product be quantitatively
separated from the reaction mixture before measurement (2,3,6-8).
The separation
methods are generally suitable for purified enzyme, but are
too difficult or time-consuming for routine use. Furthermore, gastric juice contains various substances such as
amino acids or small peptides that may interfere or increase
blank values. Other assays (2, 3, 9) have not been widely
accepted, owing to the poor availability
of the substrates
involved.
Azocoll is a commercially available complex of particulate
collagen and azo-dye (7, 10). The complex is stable in acid,
but treating it with a protease liberates the dye in a soluble
form. The unreacted azocoll can be removed by centrifugalion and the dye in the supernate measured colorimetrically.
We have studied the suitability of azocoll for measuring
pepsin activity in gastric perfusate from unstimulated rats
or samples of gastric juice from dogs in which the pepsin
secretion has been decreased pharmacologically. The use of
azocoll should simp1i
in biological fluids.
Materials and Methods
the quantification
of pepsin activity
Pharmacology II, Hofl’mann-La Roche Inc., Nutley, NJ 07110.
1 Address correspondence to this author (Bldg. 86, Room 702).
Received November 9, 1983; accepted February 23, 1984.
Secretions of canine gastric juice were collected from 12to 15-kg mongrel dogs of either sex that had been prepared
with a vagally-innervated (Amdrup)
pouch in the lesser
curvature (11). Gastric secretion in the dogs was stimulated
by intravenous infusion of histamine
dihydrochloride,
25
pg/kg of body weight per hour. Gastric juice from 200- to
350-g male Sprague-Dawley
(Charles River, strain CD) rats
was collected from a gastric fistulaas previously described
(12). Gastric juice was collected on ice and stored at -25 #{176}C
until assayed for pepsin activity.
Procedure
Azocoll pepsin assay. Azocoll (10 mg/mL) was suspended
in sodium citrate buffer, 30 mmol/L, pH 3.0. Purified pepsin
was dissolved in buffer immediately before the assay. To
start the assay, we mixed purified pepsin with 10 mg of
azocoll to give 10 mg per microgram
of enzyme or, in the
case of gastric juice, we added 1 mg of azocoll per microliter
of gastric juice. The samples were incubated at 37 #{176}C
for not
more than 20 mm. Enzyme activity was stopped by adding
0.25 volume of cold sodium phosphate buffer (1.5 moIJL, pH
7.0) and placing the assay tubes in an ice bath. The tubes
were centrifuged (1200 x g, 10 mm, 4 #{176}C).
Aliquots (typically 300 /LL) of the supernate were placed in microtiter plates
and the absorbance was measured at 520 nm with a
Dynatech Model MR600 colorimeter. Absorbances (A) were
corrected to a 1-cm pathlength as follows:
corr. (1-cm) A
=
uncorr. A x
(458 41cm)
(sample vol, jL)
The factor 458 41cm was determined by measuring the
absorbance of dye solution in a 1-cm pathlength cuvette
with a Beckman DU spectrophotometer and with the Dynatech colorimeter. Because the molar absorptivity
of the
product has not been determined, the enzyme activity is
reported in terms of the change in absorbance per minute of
CLINICAL CHEMISTRY, Vol. 30, No. 5, 1984 707
incubation per milligram of enzyme
or per milliliter
of
gastric juice.
Hemoglobin
pepsin
assay.
Pepsin activity
was measured
with hemoglobin as substrate, as described
by Rick and
Fritach (6).The tyrosine product was quantified by measuring the absorbance of the tyrosine-phenol
reagent complex
at 690 nm as described above.
pH and inhibitor experiments.
Pepstatin A was dissolved
in an equivolume solutionof ethanollwaterimmediately
before use. A 100 zg/mL solution of purified pepsin was
pretreated at 37#{176}C
for 10 mm with pepstatin, ethanol, or
buffer at the desired pH. We used the following buffers (30
mmol/L, with the pH determined at 37 #{176}C):
pH 1.2 iodate,
pH 1.9 trichioroacetate, pH 2.4 sulfate, pH 2.8 phosphate,
pH 2.8-3.3 and 4.8 citrate, pH 3.7-4.2 formate. Activity was
measured by adding 100 M.L of the pretreated enzyme to
1000 ,uL of azocoll containing the inhibitor, ethanol, or the
appropriate buffer. We stopped the reactions with 367 p.L of
cold phosphate buffer as described above and measured the
absorbances of the supernates.
Results
Optimizing Conditions for Measuring
with Azocoll
We determined
obtained at azocolllpepsin ratios exceeding >1000 g/g, or at
ratios of azocoll to dog gastric juice of 1 mg/pL. With gastric
juice from unstimulated
animals, optimum activity was
obtained at about 10-fold lower ratios (results not shown).
The results reported were obtained with 50-100
mesh
azocoll; preliminary
studies with 100-250 mesh azocoll (not
shown) suggest that the finer-mesh substrate yields less
color.
When the ratio of azocoll to enzyme or gastric juice was
maintained in the optimum range as indicated above, the
release of dye was proportional both to time, for at least 20
mm (Figure 3), and to the quantity of enzyme or gastric
juice assayed (Figure 4).
Pepsin activity is generally measured at 37 #{176}C
(4, 7, 13).
When pepsin activity was measured
at 22 and 50#{176}C
by the
azocoll method, the activity was respectively 5 and 41% of
that determined at 37#{176}C.
The reported pH optimum for pepsin varies from 1.0 to 3.5
(3,13), depending on the substrate. For azocoll, we found the
activity to be essentially the same between pH 1.2and3.4
(Figure 5). At pH >4.9 no activity could be detected.
Pepsin Activity
the optimum
ratio of azocoil to purified
enzyme activity. The
1 and 2) indicate that optimum activity is
pepsin or gastric juice for maximum
results
(Figures
0
0
4
4
a
0
C
0
N
4
0
TINE
>I-
(mIni
Fig. 3. Pepsin activity of gastric juice (0) and purified enzyme
function of timeof incubation
Each point represents a singledetermination
>
I-
I.,
4
(#{149})
as a
Ui
1n.j
2
Ui
AZOCOLL
TO PURIFIED
PEPSIN
RATIO
(gg)
Fig. 1. Effect on enzyme activity of varying the azocolVpepsinratio
Activity was measured as described in Methodsfor 20 to 30 mm at vanous ratios
of azocoll to purified enzyme. Each point represents the mean of two or more
determinations
E
0.OI
4
0
1-
5.1
I-
>
I-
U
4
UI
N
z
UI
//
0
QUANTITY
C
001
0.1
AZOCOLL
TO GASTRIC
JUICE
1.0
RATIO
(mg-L’)
Fig. 2. Effecton pepsin activity of varying the azocoll/gastricjuiceratio
Each point is the mean of two or moredetem*iationsat a singleratiofor the
serne gasthc-uIce sample
708
CLINICAL CHEMISTRY, Vol. 30, No. 5, 1984
(g
or L)
Fig. 4. Pepsin activity as a function of amount of sample
Purifiedenzyme (Mg,S) wasvaned ata fixed ratio of azocollto pepsin (3000 g/g).
The linear correlation coefficient (,) = 0.973 (n = 20) and y = 0.00129x 0.00019. Gastric juice (ML, 0) was assayed at an azocolLfju,ce ratio of 3 mg/FL.
For these data r = 0.992 (n = 17) and y = 0.00344x - 0.00024. Each point
represents the mean (with SE indicated) of three tofive determinations
Furthermore,
as reported
by Riggs and Staclie (14), the
blood,whichobviouslyinterfere
with the hemoglobin-substrate
assay, were present in some
of the gastric juice samples.
When hemoglobin was used as the substrate, pepstatin
inhibited the purified porcine pepsin with an apparent
because substances such as
choice of buffer is unimportant.
Precisionof the Azocoll Method
The within-run
precision (CV) with purifiedenzyme
is
inhibitionconstant
typically 4.4%, the between-run precision is 12.1% (Table 1).
Similar
precision
was observed with gastric juice from
histamine-stimulated
dogs or carbachol-stimulated
rats.
When the activity was reduced pharmacologically
with the
synthetic
prostaglandin
Ro 22-6923, or when the gastric
perfusate was from unstimulated rats (Table 1), the withinrun CV was 8.7 to10.1%,the between-run CV 18.1to26.4%.
Purified porcine pepsin with a specific activity of 2300
“Anson units”/mg (2.3
min’
mg’ with hemoglobin
as substrate at 37 #{176}C
and pH 2.0) gave an activity of 3.03
M520 min’
mg’ with azocoll. The activity measured with
azocoll may be converted to “Anson units” (16) by multiplying by 760.
Because no standard preparation
of purified pepsin is
available, we have elected to express the activity of gastric
fluids in experimental units, rather than to attempt to
relate the activity to the amount of enzyme present.
We believe that this precision is acceptable, because the
activity of perfusate is close to the lower limit of reliable
measurement (0.007 A520 min’
mL’
corresponds to a
M520 of 0.01 for assay of 0.1 mL of gastric perfusate with
20-mn
of 4.5 nmol/L (4); with azocoll as
(K)
substrate, pepstatin also inhibitedpurifiedporcine pepsin
with a K of 115 nmolJL (Figure 7). At 115 nmol/L we
calculated that 0.48 mol of pepstatin was bound per mole of
pepsin. Evidently the inhibition obeys the expected stoichiometry of pepstatin binding to pepsin (15).
incubation).
The mean analytical recovery of pepsin activity observed
when 2.1 jzg of purified enzyme was added to canine gastric
juice was 101% (SD 11%, n = 6). Pepsin is stable in solutions
stored frozen, or for several days at -4 #{176}C
(6).
Comparisonof Azocoll and Hemoglobinas Pepsin
Substrates
a
Hemoglobin is widely used as a substrate for pepsin (6,
13). To see whether the azocoll method was measuring the
same activity as is determined by using hemoglobin, we
compared results by the two methods for samples of canine
gastric juice. As Figure 6 indicates, the activitymeasured
with azocoll is proportional to that measured with hemoglobin, with a linear correlation coefficient of 0.61 (p < 0.001).
We believe that the correlation coefficient is relatively low
4
A
C
#{163}
I
E
A
0
E
1.
3
I
U)
(I,
C
S
>-
#{149}
2
0
-J
0
E
0’
o.
>.
E
.
0
S
A
A
I
z
4
0
-J
I-.
>
0
I-.
U
41
(Li
0
I.
o
#{149}
I
UI
0
a
z
UI
Co
C
2
3
4
2
-
3
AZOCOLL HYDROLYSIS (AA520mt.
5
mind)
Fig. 6. Correlation between the activity measured with azocoll and the
activity measured with hemoglobin
Differentsymbolsindicategastricjuice fromdifferentdogs.y = I .055x + 0.0143. r
= 0.611, n = 40
pH al 37C
Fig. 5. Pepsin activity as a function of pH
Purifiedenzyme was pretreatedat each pH for 10 mm at 37#{176}C
before azocoll
wasadded. Each pointrepresentsthe meanof threeor moredeterminations
Table 1. Precision of Measurements of Pepsin Activity In Gastric Fluids and with Purified Enzyme
WIthin run
Sample
Purified pepsin
Stimulated
canine gastric juice
Stimulated canine gastric juice
Between run
Mean
1.44
9.59
1.61
SD5
0.06
0.41
0.16
n
cv, %
Mean
4
4
4
4.4
4.3
10.1
1.41
10.73
S0
0.17
1.35
n
5
6
1.86
0.33
6
CV, %
12.1
12.6
18.0
0.040
0.005
6
11.9
0.041
0.011
6
26.4
0.068
0.006
6
8.7
0.074
0.007
5
9.0
+ Ro 22-6923, 0.5 mg/kg
Rat gastric perfusate from unstimulated rats
Gastric perfusate from rats given carbachol, 160 pg/kg
min1 mL
(Or mg’).
CLINICAL CHEMISTRY, Vol. 30, No. 5, 1984
709
Io(
-J
0
I-
z
0
U
>-
I>
5’
U
z
(1)
a(LI
a-
I hour
0.01
5 hours
PEPSTATIN
TIME OF PERFUSION
Fig. 7. Effect of pepstatinA on the activity of purified pepsin
Pepsinwas preincubatedfor 10 mm at 37 ‘C with pepstaltn A as described in
Methods.The pepstatin was indudedwith the substrateto keep the inhibitor
concentration
constant
during
the measurementof enzyme activity.Each point
representsthe mean of two or moredeterminations.
The mean controlactivity
was 1.09 (SE 0.03) #{163}4
min1 n’,g1 (n
=
4)
Use of Azocoll for Measuring Pepsin Activity in
GastricJuice
Measuring pepsin activity in gastric juice from stimulated
animals is not difficult because of the amount of enzyme
present. However, when animals are unstimulated or when
the enzyme secretion has been reduced pharmacologically,
accurate
values
may be more difficult to determine. We
examined this problem by measuring the enzyme activity in
gastric juicefrom histamine-stimulated dogs before and
after treatment with the synthetic trimethyl prostanoid Ho
22-6923, an inhibitor of pepsin secretion (17). The results
(Figure 8) indicate that the azocoll method is quite effective
at measuring the residual enzyme after such treatment. The
prostanoid Ho 22-6923inhibits pepsin secretion but does not
directly inhibit the activity of purified porcine pepsinover
the range of 10 to 100 .rmo1JL (results not shown). In a
similar experiment,pepsinactivity
couldbe measured inrat
gastric perfusate both before and after stimulation
with
carbachol (Table1).
Discussion
Our results indicate azocoll to be an excellent substrate
for measuring the activity concentration of pepsin. Under
the conditions described, activity is proportional to time and
the amount of enzyme, and isoptimalbetween pH 1.2 and
3.4 at 37 #{176}C.
Furthermore, the activity measured is sensitive
to the inhibitor pepstatin A. These observations indicate
that the procedure measures only the proteolytic activity
associated with pepsin (13), and not some other enzyme
activity that may be present in the enzyme preparation.
We found azocoll suitable for use in measuring pepsin
activity in gastric juice. The enzyme activity measured by
azocoll is proportional to the volume ofjuice present, and the
activity correlates significantly with that measured by the
hemoglobin assay. Furthermore,
the low pepsin activity in
unstimulated rat gastric perfusate or after in vivo treatment
with Ho 22-6923 can be measured.
710
CLINICAL CHEMISTRY, Vol. 30, No. 5, 1984
0.1
1.0
CONCENTRATION,mol/L
Fig.8. Effectof the prostaglandinanalog Rb 22-6923 on pepsin actMty
in dog gastricjuice
Gastric
juicewas collectedfromanAmdruppouch.Secretionwasstimulated
by
25 g of histaminedihydmchlorideper kilogramper hour. Ro 22-6923(haed
bats) was green orally (0.5 mg/kg) after 105 mm; control dogs (cpen bars)
receivedsaline. Resultsare mean ± SE for three or four animals
The azocoll method has certain advantages over other
methods. The chromogen can be measured with a conventional visible colorimeter rather than with the ultraviolet
spectrophotometer required for other assays (9, 13). Furthermore, the broad pH optimum and the ability to use a
variety ofbuffers facilitate the measurement of activity in
gastric juice or gastric perfusate, which may vary in pH or
salt content. Finally, this method, because it involves contrifugation toremove unreacted substrate, minimizes interference by particulate material in the gastric juice or
perfusate.
References
1. Ito S. Functional
gastric morphology. Chap. 17 in Physiology of
the Gastrointestinal
Tract, 1, LR Johnson, Ed., Raven Press, New
York, NY, 1981, pp 517-550.
2. Bayliss RB, Knowles JR, WybrandtGB. An aspartic acid residue
at the active site of pepsin. The isolation and sequence of the
heptapeptide. Bwchem J 113, 377-386 (1969).
3. Hunkapiller MW, Richards JH. Studies on the catalytic machathem of pepsin usinga new synthetic substrate. Biochemistry 11,
2829-2839 (1972).
4. Aoyagi T, Kunimoto S, Morishima H, et al. Effectof pepstatin on
acid proteases. J Anti biot 24, 687-694 (1971).
5. Finlay TH, Dharmgrongartama ED, Perlmann GE. Mechanism
of the gossypolinactivation of pepsinogen. J Biol Chem 248,48274833 (1973).
6. Rick W, Fritsch W-P. Pepsin (pepsin, gastricsin, pepsinogen,
uropepsinogen). In Methods of Enzymatic Analysis, HU Bergmeyer,
Ed., Academic Press, New York, NY, 1975, pp 1046-1050.
7. Tomarelli RM, Charney J, Harding ML. The use of azoalbumin
as a substrate
in the colorimetric
determination
of peptic
and
tryptic activity. J Lab Clin Med 34, 428-433 (1949).
8. Nelson WL, Ciaccio El, Hess GP. A rapid method for the
quantitative assay of proteolytic enzymes. Anal Biochem 2, 39-44
(1961).
9. Robinson LA, White IT. A spectrophotometric method for the
analysis of pepsin. Gastroenterology 58, 798-800 (1970).
10. Oakley CL, Warrack GH, van HeyningenWE. The collagenase
(a-toxin) of Cl. wekhii type A. J Pathol Bacteriol 58, 229-235
(1946).
11. Amdrup E, Ornsholt J. Pedunculated innervated fundic pouch
from lesser curvature of stomach. Scand J Gastroenterol 12, 5
(1977).
12. Gallo-Torres HE, Kuhn D, WittC.A methodforthebioassayof
antisecretory activity in theconscious rat with acute gastric fistula:
Studies with cimetidine, somatostatin, and the prostaglandin E2
analogue Ro 21-6923. J Pharinacol Methods 2, 339-355 (1979).
13. Decker LA (Ed.).
Pepsin:pepsinogen (swine stomach mucosa).
In Worthington Enzyme Manual, Worthington BiochemicalCorp.,
Freehold, NJ, 1977, pp 239-242.
14. Riggs BC, Stadie WC. A photoelectric method for the deterinination ofpeptic activity in gastric juice. J Biol Chem 150,463-470
(1943).
15. Kunimoto S, Aoyagi T, Morishima H, et al.Mechanism of
inhibition of pepsin by pepstatin. J Antibiot 25, 251-255 (1972).
16. Anson ML. The estimation of pepsin, trypsin, papain and
cathepsin with hemoglobin. J Gen Physiol 16, 79-89 (1938).
17.Gaginella T, Bertko R, Holland G, et al. Effect of a new
prostanoid, Ro 22-6923, ongastric secretion in the dog.Gastroenterology 84, 1161 (1983).
CLINICALCHEMISTRY,Vol. 30, No. 5, 1984
711